MAP4K3 regulation of cellular stress response pathways in health and disease
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Regulation of cellular metabolic status is determined by the mTORC1 complex, which senses systemic nutrient levels and intracellular nutrients. We set out to define the signaling pathways controlling autophagy activation, and discovered that MAP4K3 phosphorylation of transcription factor EB (TFEB) dictates autophagy status in the cell, and documented that MAP4K3 autophagy regulation lies upstream of mTORC1 autophagy regulation. MAP4K3 activates mTORC1 when amino acids are plentiful, but the basis for this regulation is ill-defined. During the current funding cycle, we delineated the MAP4K3 – mTORC1 signaling pathway, linking MAP4K3 activation of mTORC1 to inhibition of AMP-activated protein kinase. Our findings reveal MAP4K3 inhibition of AMPK may occur via phosphorylation of Sirtuin-1 resulting in LKB1 inactivation. To understand the scope of MAP4K3 function, we completed an interactome and phosphoproteomics analysis, and implicated MAP4K3 in regulation of the GATOR1/2 complex, which controls mTORC1 localization to the lysosome, a prerequisite step for mTORC1 activation. We also determined that MAP4K3 can localize to the nucleus, and may participate in the DNA damage response. All these findings indicate that MAP4K3 is a central node for the regulation of cellular homeostasis, serving as a nexus for cross-talk between pathways of metabolism, cell stress, and cell survival. We have begun to examine the physiological relevance of MAP4K3 phosphoregulation by deriving lines of mice with phosphoresistant and phosphomimetic amino acid substitutions at the TFEB serine residue (S3) subject to MAP4K3 phosphorylation. As mTORC1 dysregulation is implicated in cancer and neurological disease, our results suggest that one appealing therapeutic strategy for diseases of altered mTOR signaling function would be to develop drugs to inhibit MAP4K3. To achieve this goal, we performed in silico screening for small molecules that interfere with MAP4K3 open pocket dimerization, and evaluated 13 compounds in a series of secondary and tertiary assays, identifying three promising hits. In this renewal project, we will define components of the MAP4K3 amino acid sensing dependent pathway of mTORC1 activation and delineate the molecular basis for MAP4K3 regulation of mTORC1 activation, focusing on Sirtuin- 1 phosphoregulation by MAP4K3 and the nature of MAP4K3 interaction with the GATOR1/2 complex. We propose to assess MAP4K3 regulation of autophagy and cell stress by carefully characterizing phenotypes and autophagy function in TFEB S3A and S3E mice, and determining if MAP4K3 is involved in modulating DNA damage responses. We will build on our MAP4K3 inhibitor translational drug discovery work by honing in on the most promising lead compounds through structure-activity relationship generation of a compound series, coupled with an independent in silico screen and kinase inhibitor potency testing followed by a critical path of validation assays, and we will test if our lead MAP4K3 inhibitor(s) can rescue disease phenotypes in a mouse model of tuberous sclerosis complex (TSC) and in frontotemporal dementia / tauopathy Tsc1 +/- mice .
